Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. Results A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank GAG 1D4 (16.5 kDa) was isolated. Ank GAG 1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K d ~ 1 μM, and the Ank GAG 1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank GAG 1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank GAG 1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank GAG 1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank GAG 1D4-CA with the Gag assembly and budding pathway. Conclusions The resistance of Ank GAG 1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank GAG 1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.

Figure 2 Schematic construction of the ankyrin repeat library using the pHDiEx acceptor vector. (A), The mono-ankyrin microgenes werepolymerized by insertion/ligation to pHDiEx double digested by Bsm BI and Kpn I. The construction was subsequently digested by Bsp MI andrecircularised by intramolecular ligation. This resulted in the substitution of the region containing the Rep cloning sites by the ankyrin repeats.The number of repeats differed from one clone to another, and usually ranged from 2 to 6 repeats. (B), Detailed sequences of the different DNAregions. Abbreviations: T7p, T7 promoter; pLac/RBS, lactose promoter and Ribosome binding site; DsbA ss, DsbA signal sequence; St2, Strep-Tag2tag; H6, hexa-histidine tag.polyprotein H MA-CA, corresponding to the MAp17 H MA-CA or BV-H CA, and the recombinant Gag pro-6 6 6and CAp24 domains of the HIV-1 Gag precursor. The teins purified by affinity chromatography on nickel-rationale for screening our ankyrin library on the MA- sepharose column.CA target was not only to search for MA- or/and CA-binders, but also for ankyrin(s) which recognize(s) the Screening of Gag-binding ankyrins using the phage-MAp17-CAp24 hinge region, which contains the clea- display methodvage site of the viral protease (PR). His-tagged recombi- The phage-displayed library of ankyrins was amplifiednant protein H CA, which corresponded to the mature using a conventional protocol [34,35], and Gag-binders6capsid protein CAp24, was used to identify the struc- were isolated by three rounds of selection/elution fromtural domains of the Gag precursor which contained the surface-immobilized H MA-CA protein. Elution of6ankyrin-binding site. Large amounts of recombinant H MA-CA-bound phages was performed using acidic6H MA-CA and H CA proteins were produced in Sf9 buffer for the first two rounds, followed by specific ligand6 6cells infected with recombinant baculoviruses BV- elution using excess of soluble H MA-CA protein as the6

Nangola et al. Retrovirology 2012, 9:17 Page 5 of 27http://www.retrovirology.com/content/9/1/17A 1---------10----------20----------30-3 3DARPin : DXXGXTPLHLAAXXGHLEIVEVLLKZGADVNAX Library: DXXGXTPLHXAAXXGHLEIVRLLLEHGADVNAR B C Figure 3 Consensus sequence and three-dimensional model of ankyrin module. (A), Sequence comparison between the consensus DARPinrepeat motif and the repeat motif of our ankyrin library. The red letters refer to the positions of random amino acids, and blue letters representthe residues which differ from the consensus DARPin sequence. The position of the recognition site for the restriction nuclease Bsm BI isunderlined. (B), Structural model of one single ankyrin repeat motif (or module). (C), Spatial arrangement of three modules belonging to thesame ankyrin linear sequence (triple-repeat motif ankyrin molecule). The fixed structure of the repeat motif is presented as a yellow ribbon. Thevariable amino acids on the solvent-exposed surface are shown as stick pattern; their respective number in the linear sequence is indicated inpanel B.Nangola et al. Retrovirology 2012, 9:17 Page 6 of 27http://www.retrovirology.com/content/9/1/17competititor in the third round [34,35]. Phage clones Identification of the ankyrin binding domain on HIV-1were picked at random in each eluate, and tested by Gag precursorELISA for binding to H MA-CA. Only 20% of Gag-bin- The structural domain of Pr55Gag recognized by each of6GAG GAGders were found in the first eluate, whereas a significant the three Gag-binders Ank 1B8, Ank 1D4 andGAGenrichment was observed in the second and third eluates, Ank 6B4 was determined by Far Western blot analysisand ELISA. Lysates of H MA-CA-expressing Sf9 werewith 70% of Gag-binders in both. Clones which gave a 6analyzed by SDS-PAGE, and proteins transferred tosignal 5-fold over the background signal were picked inPVDF membranes. Spontaneous cleavage of H MA-CAall eluates and sequenced. All positive clones showed two 6by insect cell proteases resulted in the occurrence ofor three ankyrin repeats flanked by N-cap and C-cap.GAGThree different clones, referred to as Ank 1B8, His-tagged N-terminal domain, H MA, migrating as the6GAG GAGAnk 1D4 and Ank 6B4 and containing three mature matrix protein of the virion, MAp17 (Figure 4B;ankyrin modules each, were identified several times; they control, rightmost lane). All three Gag-binders,GAG GAG GAGwere therefore selected for further studies. Ank 1B8, Ank 1D4 and Ank 6B4, reacted withTo evaluate the specificity of our Gag-binders, an irre- H MA-CA on blot, but not with H MA (Figure 4B). This6 6levant target protein, aRep-A3, was used in lieu of indicated that the ankyrin binding site was not located inH MA-CA. Protein aRep-A3, previously described the MA domain, but in the CA domain. As expected, no6under the acronym aRep-n4-a in our previous study reaction was obtained with the control aRep-A3-binderA3[33], is an artificial alpha-helicoidal repeat protein Ank 2D3 (Figure 4B). The reactivity towards the CA(aRep) based on thermostable HEAT-like repeats, which domain was confirmed by indirect ELISA, using recombi-folds cooperatively and shows a high stability [33]. Our nant H CA protein immobilized on nickel-coated wells.6phage-displayed ankyrin library was screened on immo- Positive signals with the CA protein were detectedA3bilized aRep-A3 protein, and aRep-A3-bound clones with all three Gag-binders, but not with Ank 2D3were checked for binding specificity and sequenced. (Figure 4C). This indicated that the binding sites ofGAG GAG GAGOne ankyrin clone with a high affinity and specificity for Ank 1B8, Ank 1D4 and Ank 6B4 on H MA-CA6A3the aRep-A3 target, referred to as Ank 2D3, was used protein were all situated in the CA domain.as the irrelevant control of H MA-CA binders in the6rest of the present study. Biochemical characterization of Gag-binding ankyrinsGAGAsshowninFigure4C,Ank 1B8 reacted with H CA6with the highest apparent affinity. However, DNA sequen-Gag-ankyrin interactioncing showed several nonconservative amino acid substitu-Gag- and aRep-A3-binding ankyrins were purified, che-tions within the highly structured scaffold domain of themically biotinylated, and assayed for their capacity ofGAG GAGbinding to their specific target in vitro. Importantly, no Ank 1B8 modules, as well as in Ank 6B4. Sincechange was detected in the interaction of the three Gag- these mutations could adversely affect the ankyrin-repeatGAG GAG GAG GAG GAGbinders Ank 1B8, Ank 1D4 and Ank 6B4, and motifs, Ank 1B8 and Ank 6B4 were excluded fromA3 GAGof controlaRep-A3-binder Ank 2D3, with their respec- our next analyses, and only Ank 1D4 was selected fortive substrates, as determined by ELISA (data not further characterization. DNA sequencing revealed thatGAGshown). This indicated that biotinylation did not alter Ank 1D4 protein comprised of three ankyrin modules,their Gag- oraRep-A3-specific binding activity. each containing different types of amino acids at the sixThe degree of Gag-specificity of biotinylated assigned positions for variable residues (Figure 5A). TheGAG GAG GAG GAGAnk 1B8, Ank 1D4 and Ank 6B4 was evaluated oligo-histidine tag allowed us to purify Ank 1D4 pro-in the presence of specific or nonspecific competitors, and tein to homogeneity by using a two-step chromatographictested in ELISA using H MA-CA-coated wells. Controls procedure, (i) affinity chromatography (Figure 5B, lane 3),6A3consisted of Ank 2D3 and aRep-A3-coated wells. and (ii) gel filtration (Figure 5B, lane 2). SDS-PAGE analy-GAGCompetitors were (i) the same ankyrin protein in its sis showed that Ank 1D4 migrated with an apparentnon-biotinylated form and (ii) non-biotinylatedaRep-A3 molecular mass of 16.5 kDa (Figure 5B), consistent withGAG GAG GAGprotein. Ank 1B8, Ank 1D4, Ank 6B4 and the theoretical mass 17.9 kDa for a protein of 163 aminoA3Ank 2D3 were all competed with their respective non- acid residues.biotinylated versions, while no significant competition wasGAG GAG GAGobserved between ankyrins Ank 1B8, Ank 1D4, Mapping of the Ank 1D4 binding site on the CAGAG domainAnk 6B4 on one hand, and aRep-A3 protein on theGAG A more refined mapping of the ankyrin binding site onother hand (Figure 4A). Interestingly, Ank 1D4 showedthe CA domain was performed using carboxyterminalthe highest signal of binding to the H MA-CA target, and6deletion mutants of Gag expressed as recombinant pro-the highest competition effect was observed with non-GAG teins in baculovirus-infected cells. Gagamb276 andbiotinylated Ank 1D4(Figure 4A).Nangola et al. Retrovirology 2012, 9:17 Page 7 of 27http://www.retrovirology.com/content/9/1/17ANo inhibitor Non-biotinylated Gag binder 2.5 Biotinylated Rep-A3 binder Non-biotinylated Rep-A3 binder 2.0 1.5 1.0 0.5 0 GAG GAG GAG A3Ank 1B8 Ank 1D4 Ank6B4 Ank 2D3 H MA-CA Rep-A3 6B C2.5 H CA 62.0 BG 1.5 < H MA-CA 6 1.0 0.5 0 < H MA 6GAG GAGFigure 4 Gag-binding activity of artificial ankyrins. (A), Competition ELISA. Samples of biotinylated Gag-binders Ank 1B8, Ank 1D4 andGAG A3Ank 6B4, and of control biotinylated aRep-A3-binder Ank 2D3 were mixed with their corresponding non-biotinylated form (black bars), ormixed with irrelevant soluble target (grey bars), or mixed with buffer containing no inhibitor (white bars). Mixtures were added to H MA-CA- or6aRep-A3-coated wells, as indicated at the bottom of the panel. Bound-ankyrins were detected by addition of HRP-conjugated extravidin,followed by the TMB substrate. (B), Far Western blotting. Lysates of BV-H MA-CA-infected Sf9 cells were electrophoresed in SDS-gel, proteins6transferred to a PVDF membrane, and membrane cut into strips. Gag-binding activity was determined by incubation of the strips with theGAG GAG GAG A3different biotinylated ankyrins Ank 1B8, Ank 1D4, Ank 6B4, and Ank 2D3, as indicated on top of the strips. On the rightmost strip, therespective positions of the Gag proteins H MA-CA and H MA were determined using anti-histidine tag antibody (arrowheads). (C), Indirect6 6GAG GAGELISA.H CA was captured on nickel-coated plate, and used as substrate for binding assay of biotinylated ankyrins Ank 1B8, Ank 1D4,6GAG A3Ank 6B4, and Ank 2D3. Bound-ankyrins were quantitated as in (A). BG, background signal.Gagamb241 mutants carried an amber stop codon in 110, corresponding to positions 132-241 in the Pr55Gagthe Pr55Gag sequence at positions 276 and 241, respec- sequence of 500 amino acids.tively [36]. Both recombinant Gag proteins had in com-GAGmon the MA domain, plus 110 residues of the CA Gag-binding parameters of Ank 1D4GAGdomain for Gagamb241, and 145 of the CA The specificity and binding parameters of Ank 1D4 toGAG for Gagamb276 [36]. Ank 1D4 was found to its H MA-CA substrate were determined by microcalori-6bind to both C-truncated Gag proteins (Figure 6). This metry (ITC). Titration of increasing amounts ofGAG GAGrestricted the Ank 1D4 binding site to the N-term- Ank 1D4 protein into sample cell containing purifiedinal region of the CA domain spanning residues 1 to H MA-CA protein gave the approximate value of 1 μM6